JP2014204718A - Haemophilus influenza type iv pili - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for discriminating disease-causing H. influenzae strains from strain Rd.SOLUTION: The invention described herein relates to a Haemophilus influenzae (H. influenzae) regulon encoding type IV pili. In particular, the invention relates to type IV pili derived from nontypeable H. influenzae (NTHi) and from H. influenzae strains a, b, c, e and f. The invention provides isolated H. influenzae pilus polynucleotides and polypeptides encoded by the polynucleotides as well as polynucleotides and polypeptides encoded by the polynucleotides involved in the assembly/disassembly of the structure. The invention also relates to uses of these polynucleotides and/or polypeptides including methods for eliciting an immune response to H. influenzae and methods of treating and preventing H. influenzae infection.

Description

  Experimental work on the invention described herein was supported by grants R01 DC03915 and R01 DC005980 from NIH / NIDCD. The US government may have certain rights in the invention.

(Field of Invention)
The invention described herein relates to Haemophilus encoding type IV pili
influenza (H. influenzae) regulon. In particular, the present invention relates to non-classifiable H.264. derived from influenza (NTHi) and H. pylori. It relates to type IV pili derived from influenza strains a, b, c, e and f. The present invention relates to H.264. Provided are isolated polynucleotides of influenza pili and polypeptides encoded by the polynucleotides, as well as polynucleotides and polypeptides encoded by the construction / degradation of the structures. The present invention also provides H.264. Methods for eliciting an immune response against influenza It relates to the use of these polynucleotides and / or polypeptides, including methods of treating and preventing influenza.

(background)
The clinical term for middle ear infection is otitis media (OM). According to Non-Patent Document 1, OM is the most common reason for sick children to seek medical care and for children in the United States to receive antibiotics and general anesthetics. The statistics show that 24.5 million doctors' workplace visits were made for OM in 1990, which represents a 200% increase over what was reported in the 1980s. Although rarely related to mortality, the morbidity associated with OM is serious. Hearing loss is a common problem associated with this disease, which often affects the development of children's behavior, education and language skills (Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4). . The socio-economic impact of OM is also great, with the direct and indirect costs of diagnosing and managing OM exceeding $ 5 billion annually in the United States alone (Non-Patent Document 5).

  OM is thought to arise as a result of infectious factors, environmental factors and host genetic factors. Bacteria such as Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are the most common infectious organisms in OM. Acute OM is a disease characterized by a rapid onset of inflammation and short-term signs and symptoms in the middle ear, while chronic OM is the presence of a relatively asymptomatic fluid (or exudate) in the middle ear. The state defined by. However, in chronic OM, despite the lack of specific signs of acute infection (ie, ear pain or fever), these abnormal middle ear fluids can persist for periods exceeding 3 months. Treatment of acute OM with antibiotic therapy is common, but antibiotic-resistant bacteria are emerging. Surgical management of chronic OM involves the insertion of a middle ear cavity ventilation tube through the ear tympanic membrane while under general anesthesia in children. This procedure is common (spread rate is about 13%; Non-Patent Document 6) and is very effective in reducing pain symptoms by draining the accumulated fluid in the middle ear. Although it is invasive, it carries the risk of overlapping (Non-patent document 7; Non-patent document 6; Non-patent document 8; Non-patent document 9). Thus, there is a need for further approaches to OM management and preferably prevention.

  The development of OM vaccines is a major cause of acute OM (AOM), as evidenced by the recent approval and publication of PREVNAR® (Non-Patent Document 10), a 7-valent capsular conjugate vaccine The factor S. Most advanced on pneumoniae. PREVNAR® is very effective for invasive pneumococcal disease, but the coverage for OM has been reported to increase the number of OM cases due to serotypes not included in the vaccine It is disappointing (6% to 8%) (Non-patent document 11; Non-patent document 12; Non-patent document 13; Non-patent document 14).

  H. Influenzae is a gram-negative bacterium that plays a role in OM, as indicated above. H. Influenzae clinical isolates are each classified as either serotype "a" to "f" or non-classifiable, depending on the presence or absence of a type-specific polysaccharide capsule on the bacterium. H. A vaccine for influenza b has been developed. Like Prevnar (registered trademark), b-type H.P. Influenzae vaccines target the polysaccharide capsule of this organism, and therefore the vaccine is composed of a capsule polysaccharide bound to a protein carrier. Unclassifiable H.coli that causes about 20% of acute OM in children and predominates in chronic OM with exudate. Little progress has been made regarding vaccines for influenza (NTHi) (Non-Patent Document 15; Non-Patent Document 16; Non-Patent Document 17). NTHi also has pneumonia, sinusitis, sepsis, endocarditis, epiglottitis, pyogenic arthritis, meningitis, postpartum infection and neonatal infection, postpartum sepsis and neonatal sepsis, acute fallopianitis and chronic fallopian tubes Inflammation, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraperitoneal infection, urinary tract infection, mastoiditis, aortic transplant infection, conjunctivitis ), Brazilian purpura, subclinical bacteremia and relapse of underlying lung diseases such as chronic bronchitis, bronchiasis and cystic fibrosis. Prototype NTHi isolate is a low passage number isolate 86-028NP recovered from children with chronic OM. This strain is well characterized in vitro (Non-patent document 18; Non-patent document 19) as well as in the Chinchilla family OM model (Non-patent document 20; Non-patent document 21; Non-patent document 22). NTHi strain 86-026NP was obtained from American Type Culture Collection, 10801 University Blvd. on October 16, 2001. , Manassas, VA 20110 and assigned the registration number PTA-4774. A contig set derived from the genome of strain 86-028NP can be found in Non-Patent Document 23.

Adhesion and colony formation are It is the first stage observed in the pathogenesis of influenza. Therefore, H.I. Influenzae expresses multiple attachment factors including hemagglutinating pili attachment factor, pili attachment factor and non-pilus attachment factor (Non-patent document 24; Non-patent document 25; and Non-patent document 26). In particular, none of the described attachment factors has been previously associated with motor function. Further, H.C. Influenzae also does not express flagella associated with motility. Twitch motility is a flagella-independent form of bacterial translocation on moist surfaces, which occurs by elongation, tethering, and then contraction of polar structures known as type IV pili. Non-patent document 28; Non-patent document 28; Non-patent document 29; Non-patent document 30). Type IV pili are typically 5-7 mm in diameter, several micrometers in length, and are composed of a single protein subunit built in a helical conformation with 5 subunits per revolution (Non-Patent Document 31; Non-Patent Document 32). Type IV pilin subunits are usually 145-160 amino acids in length, which can be glycosylated or phosphorylated. There are two classes of pilin subunits (types IVa and IVb), which are the average length of the leader peptide and mature subunit in which the N-methylated amino acid occupies the N-terminal portion of the mature protein and (disulfide region) Are distinguished from each other by the average length of the D regions. Most respiratory pathogens express class IVa pilin, whereas intestinal pathogens more typically express class IVb pilin. Type IVa pili are distinguished by the presence of a highly conserved hydrophobic N-terminal methylated phenylalanine.
Type IV pilus serves as a means of rapid colonization of new surfaces. Thus, type IV pili expression is important for both attachment and biofilm formation by many bacteria (Non-Patent Document 33; Non-Patent Document 34; Non-Patent Document 35; Non-Patent Document 36), and especially Important for toxicity of Neisseria species, Moraxella bovis, Vibrio cholerae, enteropathogenic Escherichia coli and Pseudomonas aeruginosa (Non-patent document 34; Non-patent document 35; Non-patent document 37; Non-patent document 38). A biofilm is a complex tissue of bacteria that adheres to a surface through an exopolysaccharide matrix that is excreted by bacteria. This matrix encloses the bacteria and protects them from the human immune system. Non-Patent Document 39 discloses H.264. Describes biofilm formation by influenza. It is estimated that bacterial infection can be avoided or stopped by blocking the interaction between type IV pili and the human body (Patent Document 1 issued on July 31, 2001). ).
Type IV pili expression is a complex and highly regulated bacterial function. P. In aeruginosa, the biogenesis and function of type IV pili are controlled by more than 40 genes (38). To date, only a subset of the vast number of related type IV pili genes (Non-Patent Document 28; Non-Patent Document 40) have been found in several members of the HAP (Haemophilus, Actinobacillus and Pasteurella) family (Non-patent document 41; Non-patent document 42; Non-patent document 43). No influenzae isolate has been described so far. In fact, H. influenzae has been classically described as a bacterium that does not express their structure despite the presence of cryptic gene clusters in the strain Rd genome (Non-Patent Document 44) (Non-Patent Document 45; Non-Patent Document 46). ). Strain Rd is H. It is a non-encapsulated derivative of an influenzae serotype d organism (Non-Patent Document 47; Non-Patent Document 48; Non-Patent Document 49). Although strain Rd has several toxic properties, serotype d strains are generally considered to be commensals; they do not frequently cause disease (50). Therefore, the strain H. It is important to distinguish between influenza and strain Rd.

Meyer et al., US Pat. No. 6,268,171

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(Summary of the Invention)
The present invention relates to H.264. influenzae, particularly the non-classifiable H. type. influenzae (NTHi) and H.I. It relates to the type IV pili gene cluster of influenzae strains a, b, c, e and f.
For example, the present invention provides the following items.
(Item 1)
PilA polypeptide SEQ ID NO: 2, PilB polypeptide SEQ ID NO: 4, PilC polypeptide SEQ ID NO: 6, PilD polypeptide SEQ ID NO: 8, ComA polypeptide SEQ ID NO: 10, ComB polypeptide SEQ ID NO: 12, ComC polypeptide SEQ ID NO: 14, ComD Polypeptide SEQ ID NO: 16, ComE polypeptide SEQ ID NO: 18, ComF polypeptide SEQ ID NO: 20, PilF polypeptide SEQ ID NO: 22, PilA polypeptide SEQ ID NO: 26, PilA polypeptide SEQ ID NO: 26, PilA polypeptide SEQ ID NO: 28, PilA poly Peptide SEQ ID NO: 30, PilA polypeptide SEQ ID NO: 32, PilA polypeptide SEQ ID NO: 34, PilA polypeptide SEQ ID NO: 36, PilA polypeptide SEQ ID NO: 38, PilA polypeptide SEQ ID NO: 0, PiIA polypeptide SEQ ID NO: 42 or pilA any of the polypeptides SEQ ID NO: 44 comprises a nucleotide sequence that encodes one amino acid sequence, isolated polynucleotides.
(Item 2)
pilA SEQ ID NO: 1, pilB SEQ ID NO: 3, pilC SEQ ID NO: 5, pilD SEQ ID NO: 7, comA SEQ ID NO: 9, comB SEQ ID NO: 11, comC SEQ ID NO: 13, comD SEQ ID NO: 15, comE SEQ ID NO: 17, comF SEQ ID NO: 19, pilF SEQ ID NO: 21, pilA SEQ ID NO: 25, pilA SEQ ID NO: 27, pilA SEQ ID NO: 29, pilA SEQ ID NO: 31, pilA SEQ ID NO: 33, pilA SEQ ID NO: 35, pilA SEQ ID NO: 37, pilA SEQ ID NO: 39, pilA SEQ ID NO: 41 or An isolated polypeptide comprising the nucleotide sequence of any one of pilA SEQ ID NO: 43.
(Item 3)
A vector comprising the polynucleotide according to item 1 or 2.
(Item 4)
An isolated polynucleotide comprising the amino acid sequence encoded by the nucleotide sequence of item 1 or a fragment thereof.
(Item 5)
PilA polypeptide SEQ ID NO: 2, PilB polypeptide SEQ ID NO: 4, PilC polypeptide SEQ ID NO: 6, PilD polypeptide SEQ ID NO: 8, ComA polypeptide SEQ ID NO: 10, ComB polypeptide SEQ ID NO: 12, ComC polypeptide SEQ ID NO: 14, ComD Polypeptide SEQ ID NO: 16, ComE polypeptide SEQ ID NO: 18, ComF polypeptide SEQ ID NO: 20, PilF polypeptide SEQ ID NO: 22, PilA polypeptide SEQ ID NO: 26, PilA polypeptide SEQ ID NO: 26, PilA polypeptide SEQ ID NO: 28, PilA poly Peptide SEQ ID NO: 30, PilA polypeptide SEQ ID NO: 32, PilA polypeptide SEQ ID NO: 34, PilA polypeptide SEQ ID NO: 36, PilA polypeptide SEQ ID NO: 38, PilA polypeptide SEQ ID NO: 0, PilA polypeptide SEQ ID NO: 42 or PilA polypeptide SEQ ID NO: 44, or comprising the amino acid sequence of any one of peptide fragments thereof, isolated polynucleotides.
(Item 6)
6. A composition comprising the polypeptide or peptide fragment of item 5 and a pharmaceutically acceptable carrier.
(Item 7)
6. An antibody that specifically binds to the polypeptide or peptide fragment of item 5.
(Item 8)
9. A composition comprising the antibody of item 8 and a pharmaceutically acceptable carrier.
(Item 9)
A method for detecting NTHi bacteria in a biological sample comprising:
(A) contacting the polynucleotide of item 1 or a fragment thereof with a biological sample, and (b) detecting hybridization of the polynucleotide in the sample, wherein A method wherein the hybridization comprises the step of indicating the presence of NTHi bacteria.
(Item 10)
A method for detecting NTHi bacteria in a biological sample comprising:
(A) contacting the polynucleotide of item 2 or a fragment thereof with a biological sample, and (b) detecting hybridization of the polynucleotide in the sample, wherein A method wherein the hybridization comprises the step of indicating the presence of NTHi bacteria.
(Item 11)
A method for detecting NTHi bacteria in a biological sample comprising:
(A) contacting the antibody of item 7 with a biological sample; and (b) detecting binding of the antibody in the sample, wherein binding is the presence of NTHi bacteria. A method comprising the steps of:
(Item 12)
Item 12. The method according to any one of Items 9, 10 or 11, wherein the biological sample is selected from the group consisting of serum, sputum, ear fluid, blood, urine, lymph, and cerebrospinal fluid. ,Method.
(Item 13)
A method for inducing an immune response against an NTHi bacterium, wherein the method provides an immunogenic dose of one or more polypeptides or peptide fragments according to item 5 to a patient at risk of NTHi bacterium infection. Administering a step of administering.
(Item 14)
A method for inducing an immune response against NTHi bacteria comprising administering an immunogenic dose of one or more polynucleotides according to item 1 to a patient at risk of NTHi bacterial infection. Including the method.
(Item 15)
Item 15. The method according to Item 14, wherein the polynucleotide is present in a plasmid or a viral vector.
(Item 16)
A method for treating or preventing an NTHi bacterial infection, the method comprising administering to a patient in need thereof a molecule that inhibits the expression or activity of the polypeptide of item 5.
(Item 17)
The method of item 16, wherein the molecule administered to the patient in need is an antisense oligonucleotide.
(Item 18)
The method according to item 16, wherein the molecule administered to a patient in need is an antibody.
(Item 19)
The method of item 16, wherein the molecule administered to the patient in need is a small molecule.
(Item 20)
Item 17. The method according to Item 16, wherein the NTHi infection is present in the middle ear.

(Polynucleotide and polypeptide of the present invention)
The present invention relates to H.264. Influenzae polynucleotides and in particular open regenerative frames derived from regulon arranged in one other gene in addition to two gene clusters. This regulon is a previously uncharacterized H. pylori. It contains the gene (pilA) encoding the major subunit of influenza type IV pili. This regulon is a gene cluster encoding the pilin polypeptides PilA (major pilin subunit), PilD (leader peptidase), PilB and PilC (involved in the construction / degradation of pilin structures); ComA, ComB, ComC, ComD From another gene cluster encoding ComE and ComF (which is involved in the capacity for transformation and pili expression); and a gene encoding PilF (required for type IV pili development) Polynucleotides are included (Watson et al., Gene, 49:56, 1996). In one embodiment, the pilus regulon is NTHi H. Influenzae strain 86-028NP.

  A polynucleotide encoding the NTHi86-028NP pilin polypeptide shown in the following SEQ ID NOs is provided by the present invention: a PilA polypeptide in SEQ ID NO: 2, a PilB polypeptide in SEQ ID NO: 4, a PilC polypeptide in SEQ ID NO: 6 PilD polypeptide in SEQ ID NO: 8, ComA polypeptide in SEQ ID NO: 10, ComB polypeptide in SEQ ID NO: 12, ComC polypeptide in SEQ ID NO: 14, ComD polypeptide in SEQ ID NO: 16, ComE polypeptide in SEQ ID NO: 18, sequence The ComF polypeptide at number 20 and the PilF polypeptide at SEQ ID NO: 22. Accordingly, alternative codon usage is specifically contemplated by the present invention. In one embodiment, the polynucleotide comprises the NTHi86-028NP gene sequence set forth in SEQ ID NO: 1 that encodes each of the aforementioned polypeptides: pilA in SEQ ID NO: 1, pilB in SEQ ID NO: 3, in SEQ ID NO: 5 pilC, pilD in SEQ ID NO: 7, comA in SEQ ID NO: 9, comB in SEQ ID NO: 11, comC in SEQ ID NO: 13, comD in SEQ ID NO: 15, comE in SEQ ID NO: 17, comF in SEQ ID NO: 19; and pilF in SEQ ID NO: 21 . Each of the above polynucleotide sequences includes a final three nucleotides representing a stop codon.

  Polynucleotides encoding PilA polypeptides derived from NTHi clinical isolates 1728 MEE, 1729 MEE, 3224 A, 10548 MEE, 1060 MEE, 1885 MEE, 1714 MEE, 1236 MEE, 1128 MEE and 214 NP are also provided. The amino acid sequences of these PilA polypeptides are shown in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44, respectively. Furthermore, the possibility of alternative codon usage is specifically contemplated in the polynucleotide encoding the polypeptide. In one embodiment, the polypeptide is encoded by the nucleotide sequences shown in SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43, respectively.

  The present invention relates to (a) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35 under stringent conditions. The complement of the nucleotide sequence shown in 37, 39, 41 or 43; (b) a polynucleotide which is an allelic variant of any of the polynucleotides listed above; (c) of any of the protein species listed above; A polynucleotide that encodes a homolog; or (d) a polynucleotide that hybridizes to a polynucleotide that encodes a polypeptide comprising a particular domain or truncation of a polypeptide of the invention. Other non-classifiable types from influenzae strains and H. pneumoniae. Specifically contemplated are type IV pilin polynucleotides from influenzae strains a, b, c, e and f. These polynucleotides are used as probes or primers respectively as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37, It can be identified and isolated by standard techniques in the art such as hybridization and polymerase chain reaction using some or all of the 39, 41 or 43 polynucleotides.

  The polynucleotides of the invention also include nucleotide sequences that are substantially equivalent to the polynucleotides listed above. The polynucleotide according to the invention is, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84% relative to the NTHi polynucleotides listed above. %, At least 85%, at least 86%, at least 87%, at least 88% or at least 89%, more typically at least 90%, at least 91%, at least 92%, at least 93% or at least 94% and even more representative Specifically, it may have at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

  Included within the scope of the nucleic acid sequences of the present invention are those under stringent conditions, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29 , 31, 33, 35, 37, 39, 41 or 43, or a nucleic acid sequence fragment that hybridizes to the complement thereof, which fragment is greater than about 5 nucleotides, preferably greater than 7 nucleotides. More preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. For example, 15 nucleotides, 17 nucleotides, or 20 nucleotides or larger fragments that are selected (ie, specifically hybridize to any one of the polynucleotides of the invention) are contemplated. These nucleic acid sequence fragments that can specifically hybridize to the NTHi polynucleotides of the invention can be used as probes to detect the NTHi polynucleotides of the invention and / or other NTHi polynucleotides of the invention and other It can be distinguished from bacterial genes and is preferably based on a unique nucleotide sequence.

  The term “stringent” is used herein to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and concentration of denaturing agents (eg, formamide). Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride at 65 ° C. to 68 ° C., 0.0015 M sodium citrate or 0.015 M sodium chloride at 42 ° C., 0.0015 M sodium citrate, and 50% formamide. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989).

  More stringent conditions (eg, higher temperature, lower ionic strength, higher concentrations of formamide, or other denaturing agents) can also be used, but the rate of hybridization is affected. In cases where deoxyoligonucleotide hybridization is involved, additional exemplary stringent hybridization conditions are: 37 ° C. (for 14 base oligos), 48 ° C. (for 17 base oligos), 55 ° C. (20 bases). For oligos), and at 60 ° C. (for 23 base oligos) in 6 × SSC 0.05% sodium pyrophosphate.

Other factors can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl - pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO _4, (SDS), ficoll, Denhardt's solution (Denhardt's solution), sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate, but other suitable factors can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4, however, under typical ionic strength conditions, the rate of hybridization is almost independent of pH. Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, see IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and to allow different sequence related DNAs to form hybrids.

  As indicated above, the polynucleotides contemplated by the present invention are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33. , 35, 37, 39, 41 or 43, but also include, for example, allelic variations and species variations thereof. Allelic variation and species variation are SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, 37, 39, 41 or 43, preferably open reading frames therein, representative fragments thereof, or SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 25, 27, 29, 31 , 33, 35, 37, 39, 41 or 43, a sequence provided in a nucleotide sequence that is at least 90% identical, preferably 95% identical to an open reading frame, in another isolation of the same or another species It can be routinely determined by comparison with a body-derived sequence. Preferred computer program methods for determining identity and similarity between two sequences include GAP (Devereux et al., Nucl. Acid. Res., 12: 387, 1984; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215: 403-410, 1990), but are not limited to these. The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda, MD 20894; Altschul et al., Supra). The well-known Smith-Waterman algorithm can also be used to determine identity.

  The polynucleotides of the present invention can be isolated from natural sources or can be isolated from standard chemical techniques (eg, the phosphotriester method described in Matteucci et al., J Am Chem Soc. 103: 3185 (1981)). ).

  An antisense polynucleotide complementary to a polynucleotide encoding a pili polypeptide of the invention is also provided.

The polypeptides of the present invention include the pilin polypeptides PilA, PilD, PilB, PilC, ComA, ComB, ComC, ComD, ComE, ComF and PilF. In one embodiment, the polypeptide includes NTHi as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 22, respectively.
The 86-028NP amino acid sequence is mentioned. Polypeptides of the invention also include PilA polypeptides set forth in SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44. In a further embodiment, the type IV pilin polypeptide of the invention is a non-classifiable H. coli. type IV pilin polypeptides of H. influenzae strains and H. coli. It is a type IV pilin polypeptide derived from influenzae strains a, b, c, e and f.

  The polypeptides of the present invention specifically include peptide fragments (ie, peptides) that retain one or more biological or immunogenic properties of the full-length polypeptides of the present invention. In one embodiment, the PilA peptide fragments provided by the invention are named TfpQ2, TFPQ3, TfpQ4 and OLP3, amino acids 35-68 of SEQ ID NO: 2, amino acids 69-102 of SEQ ID NO: 2, respectively. It includes amino acids 103 to 137 of SEQ ID NO: 2 and amino acids 21 to 35 of SEQ ID NO: 2.

  The invention also provides polypeptides having one or more conservative amino acid substitutions that do not affect the biological and / or immunogenic activity of the polypeptide. Alternatively, the polypeptides of the invention are contemplated to have conservative amino acid substitutions that may or may not alter biological activity. The term “conservative amino acid substitution” refers to non-original residues (including naturally occurring and non-naturally occurring amino acids) that have little or no effect on the polarity or charge of the amino acid residue at that position. The substitution of the original amino acid residue by For example, a conservative substitution results from the replacement of a nonpolar residue in a polypeptide with any other nonpolar residue. In addition, any original residue in the polypeptide can also be replaced by alanine according to the method of “alanine scanning mutagenesis”. Naturally occurring amino acids are characterized on the basis of their side chains as follows: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; uncharged polarity: glutamine, asparagine, serine, threonine, Tyrosine; and nonpolar: phenylalanine, tryptophan, cysteine, glycine, alanine, valine, proline, methionine, leucine, norleucine, isoleucine. The general rules for amino acid substitution are shown in Table 1 below.

本発明はまた、生物学的活性および／または免疫原性活性を保持する、本発明のポリペプチドの改変体（例えば、配列番号２、４、６、８、１０、１２、１４、１６、１８、２０または２２のポリペプチドに対して、少なくとも約６５％、少なくとも約７０％、少なくとも約７５％、少なくとも約８０％、少なくとも約８５％、少なくとも約８６％、少なくとも約８７％、少なくとも約８８％、少なくとも約８９％、少なくとも約９０％、少なくとも約９１％、少なくとも約９２％、少なくとも約９３％、少なくとも約９４％、代表的には少なくとも約９５％、少なくとも約９６％、少なくとも約９７％、より代表的には少なくとも約９８％、または最も代表的には少なくとも約９９％のアミノ酸同一性を示すポリペプチド）を提供する。

The invention also provides variants of the polypeptides of the invention (eg, SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 that retain biological and / or immunogenic activity. , 20 or 22 polypeptides, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88% At least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, typically at least about 95%, at least about 96%, at least about 97%, More typically, polypeptides that exhibit at least about 98%, or most typically at least about 99% amino acid identity).

  The present invention contemplates that the polynucleotide of the present invention may be inserted into a vector for amplification or expression. For expression, the polynucleotide is operably linked to appropriate expression control sequences (eg, a promoter sequence and a polyadenylation signal sequence). Further provided is a host cell comprising a polynucleotide of the invention. Exemplary prokaryotic host cells include E. coli. Examples include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella and Serratia. Specifically contemplated are methods of producing a polypeptide of the invention by growing the host cell and isolating the polypeptide from the host cell or growth medium. One or more polynucleotides from the pili of the pilus regulon can be expressed in the host cell. For example, both the expression of the pilA gene alone and the expression of multiple polynucleotides from the pili pilus regulon are specifically contemplated to affect the construction of the native pili structure. Alternatively, the polypeptides of the invention can be prepared by chemical synthesis using standard means. Particularly convenient is the solid phase technique (see, eg, Erikson et al., The Proteins (1976) v. 2, Academic Press, New York, p. 255). Automatic solid phase synthesizers are commercially available. Furthermore, sequence modifications are easily made by substitution, addition or deletion of appropriate residues. For example, a cysteine residue can be added to the carboxy terminus to provide a sulfhydryl group for convenient attachment to a carrier protein, or a spacer element (eg, an additional glycine residue) can be added to the C-terminus. Can be incorporated into the sequence between the amino acid linked in and the rest of the peptide.

  The term “isolated” refers to material that has been removed from other components of the environment in which the substance is originally present and that is essentially free of other components of the environment in which the substance is originally present. For example, the polypeptide is separated from other cellular proteins, or the DNA is separated from other DNA adjacent to the DNA in the genome in which the DNA originally resides.

(antibody)
The present invention relates to the H.264 of the present invention. Antibodies are provided that bind to an antigenic epitope that is unique (ie, specific) to an influenzae pili polypeptide. Multiple H.P. Antibodies that bind to antigenic epitopes that are common among influenza subtypes but are unique with respect to any other antigenic epitope are also provided. This antibody can be a polyclonal antibody, a monoclonal antibody, antibody fragments that retain their ability to bind to their unique epitopes (eg, Fv fragments, Fab fragments and F (ab) 2 fragments), single chain antibodies and human antibodies Or it may be a humanized antibody. Antibodies can be produced by standard techniques in the art using host cells expressing a pili polypeptide of the invention or a pilin polypeptide of the invention as an antigen.

  The present invention provides antibodies specific for the pilin polypeptides of the present invention and fragments thereof, It shows both the ability to kill influenzae bacteria and the ability to protect humans from infection. The present invention also provides an antibody specific for the polypeptide of the present invention, which reduces toxicity, inhibits adhesion, inhibits biofilm formation, inhibits twitch motility, cell division And / or H. inhibits the entry of influenzae bacteria into the epithelium and / or Enhances phagocytosis of influenza bacteria.

  An in vitro complement-mediated bactericidal assay system (Musher et al., Infect. Immun. 39: 297-304, 1983; Anderson et al., J. Clin. Invest. 51: 31-38, 1972) Can be used to measure the bactericidal activity of

  H. of the present invention. It is also possible to confer short-term protection to the host by passive immunotherapy through the administration of preformed antibodies against the influenzaza polypeptide. Thus, the antibodies of the invention can be used in passive immunotherapy. Human immunoglobulins are preferred in human medicine because heterologous immunoglobulins can elicit an immune response against their foreign immunogenic components. Such passive immunization can be used in emergencies for immediate protection of non-immunized individuals who are at special risk.

  In another embodiment, the antibodies of the invention can be used in the production of anti-idiotypic antibodies, which can then be used as antigens to stimulate an immune response against a pilin epitope.

(Method and composition for eliciting an immune response)
The present invention relates to one or more H.P. A method for inducing an immune response in an individual against an influenza type IV pili polypeptide is contemplated. In certain embodiments, the method elicits an immune response against the PilA protein. These methods include immune responses that inhibit bacterial replication, H. an immune response that blocks attachment of influenzae; Elicit one or more immune responses including, but not limited to, immune responses that prevent influenza constriction and biofilm formation. In one embodiment, the method includes administering an immunogenic dose of a composition comprising one or more polypeptides of the invention. In another embodiment, the method includes the step of administering an immunogenic dose of a composition comprising cells that express one or more polypeptides of the invention. In yet another embodiment, the method comprises administering an immunogenic dose of a composition comprising one or more polynucleotides encoding one or more polypeptides of the invention. The polynucleotide can be a naked polynucleotide that is not associated with any other nucleic acid, or a vector (eg, a plasmid vector or a viral vector (eg, an adeno-associated viral vector or an adenoviral vector)). Can exist in. This method can be used in combination in a single individual. This method involves the H.H. It can be used before or after infection with influenza.

  In one embodiment of the method of the invention, the composition of the invention is administered as an initial immunization dose, followed by one or more booster doses. Co-administration of proteins or polypeptides that advantageously enhances the immune response (eg, cytokines (eg, IL-2, IL-12, GM-CSF), cytokine-inducing molecules (eg, Leaf) or costimulatory molecules) also Intended.

  An “immunogenic dose” of a composition of the invention is a humoral immunity that is detectable after administration as compared to a detectable immune response before administration or compared to a standard immune response before administration. A response (antibody) and / or a cellular immune response (T cell) is generated. The present invention contemplates that the immune response resulting from the above methods can be protective and / or therapeutic. In a preferred embodiment, the antibody and / or T cell immune response causes the individual to H.264. Protects against influenza infection, particularly infection of the middle ear and / or nasopharyngeal or lower respiratory tract. In this use, the exact dose will depend on the patient's health and weight, mode of administration, the nature of the formulation, etc., but generally will be from about 1.0 μg to about 5000 μg, more commonly per 70 kg patient. Specifically, it is in the range of about 10 μg to about 500 μg per 70 kg body weight.

  A humoral immune response can be measured by a number of well-known methods, such as one-way radioimmunodiffusion (SRID), enzyme immunization (EIA), and hemagglutination inhibition (HAI). In particular, SRID utilizes a layer of gel (eg, agarose) that contains the immunogen to be tested. The gel is cut into wells and the serum to be tested is placed in the wells. Diffusion of the antibody out into the gel leads to the formation of a precipitating ring whose area is proportional to the concentration of antibody in the serum being tested. EIA is also known as ELISA (Enzyme Linked Immunoassay) and is used to measure total antibody in a sample. The immunogen is adsorbed on the surface of the microtiter plate. The test serum is exposed to the plate and subsequently exposed to an enzyme conjugated immunoglobulin (eg, IgG). The enzyme activity attached to the plate is quantified by any convenient means (eg, spectrophotometry) and is proportional to the concentration of antibody to the immunogen present in the test sample. HAI takes advantage of the ability of an immunogen (eg, a viral protein) to aggregate chicken erythrocytes (such as). This assay is a neutralizing antibody (ie, an antibody that can inhibit hemagglutination). Test serum dilutions are incubated with a standard concentration of immunogen, followed by the addition of red blood cells. The presence of neutralizing antibodies inhibits red blood cell aggregation by the immunogen. Tests for measuring cellular immune responses include determining late-type hypersensitivity or measuring the proliferative response of lymphocytes to a target immunogen.

  The present invention also provides compositions suitable for eliciting an immune response against the pili polypeptide of the present invention. As indicated above, the composition comprises one or more pilus polypeptides, one or more cells expressing one or more polypeptides, or one or more encoding a one or more pilus polypeptides. Including polynucleotides. The composition can also include other ingredients such as carriers and adjuvants.

  In the composition of the present invention, the pili polypeptide can be fused with another protein when produced by recombinant methods. In one embodiment, the other protein alone may not elicit antibodies, but it forms a fusion protein that stabilizes the first protein and maintains immunogenic activity. In another embodiment, the fusion protein is another protein that is immunogenic (eg, glutathione-S-transferase (GST) or β-galactosidase), solubilizing the fusion protein to facilitate its production and purification. A relatively large coprotein. Other proteins can act as adjuvants in the sense of providing systemic stimulation of the immune system. Other proteins can be fused to either the amino terminus or the carboxy terminus of the NTHi protein of the invention.

  In other compositions of the invention, the pili polypeptide may be bound to the carrier material in other ways. Any method of making such bonds known in the art can be used. Coupling is a heterobifunctional factor that produces a disulfide bond at the end of one functional group and a peptide bond at the end of another functional group (eg, a disulfide amide-forming factor (eg, N-succimidimid-3- (2-pyridyl) Dithio) propionate (SP-)) (see, for example, Jansen et al., Immun. Rev. 62: 185, 1982) and thioether linkages rather than disulfide linkages. A bifunctional coupling agent to form (eg, reactive esters such as 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4- (N-maleimido-methyl) cyclohexane-1-carboxylic acid), and Carboxyl And succinimide or 1-hydroxy-2-nitro-4-sulfonic acid (for example, succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate (SMCC) as a sodium salt) It can be formed using a coupling agent that activates the group.

  The pili polypeptide can be formulated as a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino group of the peptide), which can be inorganic acids (eg, hydrochloric acid or phosphoric acid) or organic acids (eg, acetic acid, (Oxalic acid, tartaric acid, mandelic acid). Salts formed with free carboxyl groups can also contain inorganic bases (eg, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide), and organic bases (eg, isopropylamine). , Trimethylamine, 2-ethylaminoethanol, histidine, and procaine).

  The composition of the present invention may further comprise an adjuvant. For example, known adjuvants include emulsions (eg, Freund's adjuvant and other oil emulsions), purified saponins from Bordetella pertussis, MF59, Quillaja saponaria (QS21), aluminum salts (eg, hydroxide, phosphate) Salts and alum), calcium phosphate (and other metal salts), gels (eg, aluminum hydroxide salts), mycobacterial products including muramyl dipeptide, solids, particles (eg, liposomes and virosomes). Examples of natural and bacterial products known to be used as adjuvants include monophosphoryl lipid A (MPL), RC-529 (synthetic MPL-like acylated monosaccharide), E. coli. OM-174, a holy toxin (eg, cholera toxin (CT) or one of its derivatives, pertussis toxin (PT) and E. coli heat labile toxin (LT) or its derivatives 1), and CpG oligonucleotides. Adjuvant activity can be affected by a number of factors, such as carrier effects, reservoir formation, altered lymphocyte recirculation, T lymphocyte stimulation, direct B lymphocyte stimulation and macrophage stimulation.

  The compositions of the present invention are typically formulated as injectables, either as liquid solutions or suspensions; solids suitable for dissolution or suspension in liquid prior to injection can also be prepared. This preparation can also be emulsified. The active immunogenic component is often mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccine. Vaccines are routinely administered parenterally, by injection (eg, either subcutaneously or intramuscularly).

  Additional formulations suitable for other modes of administration include suppositories and, in some cases, oral formulations. With respect to suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories are 0.5% to 10%, preferably 1% to 2%. It can be formed from a mixture containing active ingredients in a range. Oral formulations include commonly used excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, 10% to 95% active ingredient, preferably 25% to 70% active ingredient including.

  The compositions also include jet injectors, microneedles, electroporation, sonoporation, microencapsulation, polymers or liposomes, transdermal routes, transmucosal routes and intranasal routes using nebulizers, aerosols and nasal sprays. Can be administered via Natural or synthetic polymers (eg starch, alginate and chitosan, D-poly L-lactate (PLA), D-poly DL-lactic-coglycolic microspheres, polycaprolactone, polyorthoesters, polyanhydrides and polyphosphazenes Microencapsulation with polyphosphazanes is useful for both transdermal and transmucosal administration. Polymer conjugates comprising synthetic polyornitate, poly-lysine and poly-arginine or amphiphilic peptides are useful for transdermal delivery systems. Furthermore, due to their amphiphilic nature, liposomes are contemplated for transdermal, transmucosal and intranasal vaccine delivery systems. Common lipids used for vaccine delivery include N- (1) 2,3- (dioleoyl-dihydroxypropyl) -N, N, N-trimethylammonium-methyl sulfate (DOTAP), dioleyloxy -Propyl-trimethylammonium chloride (DOTMA), dimystoxypropyl-3-dimethyl-hydroxyethylammonium (DMRIE), dimethyldioctadecylammonium bromide (DDAB) and 9N (N ', N-dimethylaminoethane) carbamoyl) cholesterol (DC-Chol). The combination of helper lipids and liposomes enhances liposome uptake through the skin. These helper lipids include dioleoylphosphatidylethanolamine (DOPE), dilauroyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylethanolamine (DMPE), and dipalmitoyl phosphatidylethanolamine (DPPE). In addition, triterpenoid glycosides or saponins and chitosan (deacetylated titanium) from Chile soap tree bark (Quillaja saponaria) are contemplated as useful adjuvants for intranasal and transmucosal vaccine delivery. ing.

  Formulations can be in unit dose containers or multi-dose containers (eg, sealed ampoules and vials) and stored in a lyophilized state requiring only the addition of a sterile liquid carrier just prior to use. Can be done.

(Method of inhibiting H. influenzae)
Alternatively, the present invention provides for H.P. Methods of inhibiting influenza type IV pili function are included. This method may include, for example, one or more antibodies of the invention; one or more polypeptides of the invention; one or more antisense polynucleotides of the invention; in an amount that inhibits the function of pili. Administering the above RNAi molecule; and / or one or more small molecules to an individual. In vitro assays can be used to demonstrate the ability to inhibit pili function. Embodiments of these methods include, for example, inhibitors of pilus polypeptide synthesis and / or pilus assembly, inhibitors of attachment mediated through type IV pilus, type IV pilus Inhibitors that disrupt existing biofilms, and methods that use inhibitors of twitch contraction.

  Inhibition is described in any pathological condition involved in influenzae (eg, OM, pneumonia, sinusitis, sepsis, endocarditis, epiglottitis, purulent arthritis, meningitis, postpartum and neonatal infections, postpartum sepsis and Neonatal sepsis, acute and chronic tubal inflammation, epiglottis, pericarditis, cellulitis, osteomyelitis, endocarditis, cholecystitis, intraperitoneal infection, urinary tract infection, mastoiditis, aortic transplantation Contemplated for infection, conjunctivitis, Brazilian purpura, subclinical bacteremia and relapse of underlying lung disease (eg, chronic bronchitis, bronchiectasis and cystic fibrosis).

H. Compositions comprising inhibitors of influenza type IV pili function are provided. This composition may consist of only one of the aforementioned active ingredients, may comprise a combination of the aforementioned active ingredients, or may comprise further active ingredients used to treat bacterial infections. Good. As discussed above, the composition can include one or more additional ingredients (eg, a pharmaceutically effective carrier). Also, as discussed above, the dosage and frequency of administration of this composition is determined by standard techniques and depends on, for example, the individual's weight and age, the route of administration, and the severity of the symptoms . Administration of the pharmaceutical composition can be by standard routes in the art (eg, parenteral, intravenous, oral, buccal, nasal, pulmonary, rectal, intranasal, or intravaginal).

(Animal model)
The method of the present invention can be demonstrated in a chinchilla model that has been widely accepted as an experimental model for OM. In particular, the chinchilla model of NTHi-induced OM has been well characterized (Bakalettz et al., J. Infect. Dis., 168: 865-872, 1993; Bakaletz and Holmes, Clin. Diagnostic. Lab. Immunol., 4 : 223-225, 1997; Suzuki and Bakaletz, Infect.Immun., 62: 1710-1718, 1994; Mason et al., Infect.Immun., 71: 3454-3462, 2003), several NTHi outer membrane proteins for OM. , Outer membrane protein combinations, chimeric synthetic peptide vaccine components, and adjuvant formulations have been used to determine the prophylactic efficacy (Bakaletz et al., Vaccine, 15: 955-9). 1, 1997; Bakaletz et al., Infect.Immun., 67: 2746-2762, 1999; Kennedy et al., Infect.Immun., 68: 2756-2765, 2000; Kyd et al., Infect.Immun., 66: 2272-2278, 2003; Novotny and Bakaletz, J. Immunol., 171, 1978-1983, 2003).

In this model, adenovirus causes chinchilla to be transformed into H. coli. Susceptible to influenzae-induced OM, which allowed the establishment of appropriate cell, tissue and organ culture systems for the biological assessment of NTHi (Bakaletz et al., J. Infect. Dis., 168: 865-72). 1993; Suzuki et al., Infect. Immunity 62: 1710-8, 1994). A single adenoviral infection has been used to assess leakage of induced serum antibodies into the tympanic membrane (Bakaletz et al., Clin. Diagnostic Lab Immunol., 4 (2): 223-5, 1997), and It has several active and passive immunizations targeting co-pathogens with NTHi, various NTHi outer membrane proteins, combinations of OMPs, chimeric synthetic peptide vaccine components, and adjuvant formulations as sapling seeds for otitis media Used to determine the prophylactic efficacy of the regimen (Bakaletz et al., Infect Immunity, 67 (6): 2746-62, 1999; Kennedy et al., Infect. Immun., 68 (5): 2756-65, 2000; Novotn Al, Infect Immunity 68 (4): 2119-28,2000; Poolman et al., Vaccine 19 (Suppl 1): S108-15,2000).
(Method for detecting H. influenzae bacteria)
Also provided by the present invention is a method for detecting bacteria in an individual. In one embodiment, the method includes detecting a pili of the invention in a biological sample using a primer or probe that specifically binds to the polynucleotide. Detection of this polynucleotide can be accomplished by a number of conventional techniques in the art, including, for example, hybridization and / or PCR. In another embodiment, the method comprises detecting an inventive pili polypeptide of the invention in a biological sample using an antibody of the invention that specifically binds to the polypeptide. This antibody can be any immunoassay system known in the art (radioimmunoassay, ELISA assay, sandwich assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination reaction assay, fluorescent immunoassay) , Immunological assays, protein A immunoassays, and immunoelectrophoretic assays). Biological samples utilized in this method include, but are not limited to, blood, serum, ear fluid, spinal fluid, sputum, urine, lymph fluid, and cerebrospinal fluid.

FIG. 1 shows NTHi Rd, 86-028NP (SEQ ID NO: 2), 1728 MEE (SEQ ID NO: 26), 1729 MEE (SEQ ID NO: 28), 3224A (SEQ ID NO: 30), 10548 MEE (SEQ ID NO: 32), 1060 MEE (SEQ ID NO: 34). , 1885 MEE (SEQ ID NO: 36), 1714 MEE (SEQ ID NO: 38), 1236 MEE (SEQ ID NO: 40), 1128 MEE (SEQ ID NO: 42), 214 NP (SEQ ID NO: 44) amino acid sequence alignment of the PilA polypeptide.

(Detailed description of the invention)
The following examples illustrate the invention, where Example 1 shows the sequence of the NTHi strain 86-028NP type IV pili gene of the invention and 13 clinical H. coli strains. Detection of the pilA gene in influenzae isolates is described, Example 2 shows classical type IV pili-dependent aggregate formation by NTHi strain 86-028NP, and Example 3 shows NTHi strain 86 Shows twitch motility in -028NP, Example 4 describes the observation of type IV pili on NTHi strain 86-928NP by negative staining and transmission electron microscopy, Example 5 shows the pilA mutant Example 6 describes experiments in a chinchilla model of infection with NTHi and the pilA mutant NTHi, and Example 7 describes the pilA gene from 10 NTHi clinical isolates. Example 8 describes an experiment showing an immune response to NTHi pili in children, and Example 9 is for use as an immunogen. It describes the identification of NTHi peptide fragments fit.

Example 1
Type IV pili regulon was identified in OM isolates of NTHi.

  Many strains of NTHi, including strain 86-028NP, do not possess the hif locus required for expression of hemagglutinating LKP pili (Mhlanga-Mutangadura et al., J. Bacteriol., 180 (17), 4693-4703, 1988) mostly form biofilms. Since pili are important for biofilm formation in other bacterial systems, the results of genome sequencing of NTHi strain 86-028NP (http://www.microbiological-pathogenesis.org or co-owned US patent applications) 60 / 453,134) was analyzed for genes that might encode another pili type. The data set is www. Pseudomonas. com, including PilQ and PilT. BLASTed using the tblasn algorithm with default parameters using the Pseudomonas aeruginosa protein annotated as type IV pili or a twitch motility association for aeruginosa. P. Polypeptides translated for the multicida PilA protein were also used in this search (Doughty et al., Vet. Microbiol., 72, 79-90, 2000).

What initially appeared to be a latent type IV pili locus was identified. Specifically, in strain 86-028NP, influenzae strain Rd, A. pleuropneumoniae and P. There are four genes that are highly homologous to those in Multocida (Stevenson et al., Vet. Microbiol., 92, 121-134, 2003; Doughty et al., supra; Zhang et al., FEMS.
Microbiol Lett, 189, 15-18, 2000; and Rufolo et al., Infect. Immun. 65, 339-343, 1997). These genes encode PilA, PilB, PilC and PilD in strain Rd (Dougherty and Smith, Microbiology, 145 (2), 401-409, 1999).

  The NTHi strain 86-028NP regulon is intended to be involved in the construction / degradation of the pilin structure, pilin polypeptide PilA (major pilin subunit), PilD (leader peptidase), PilB and PilC (transcribed from the same mRNA). A gene cluster of polynucleotides encoding the pilin polypeptides ComA, ComB, ComC, ComD, ComE and ComF (which are involved in the capacity for transformation and pili expression); and Contains another gene encoding PilF (required for type IV pili development). The amino acid sequence of the pilin polypeptide is shown in the following SEQ ID NOs: PilA in SEQ ID NO: 2, PilB in SEQ ID NO: 4, PilC in SEQ ID NO: 6, PilD in SEQ ID NO: 8, ComA in SEQ ID NO: 10, ComB in SEQ ID NO: 12 ComC in SEQ ID NO: 14, ComD in SEQ ID NO: 16, ComE in SEQ ID NO: 18, ComF in SEQ ID NO: 20, and PilF in SEQ ID NO: 22. The gene sequences encoding the above polypeptides are shown in the following SEQ ID NOs encoding each of the aforementioned polypeptides: pilA in SEQ ID NO: 1, pilB in SEQ ID NO: 3, pilC in SEQ ID NO: 5, pilD in SEQ ID NO: 7, sequence ComA in number 9, comB in sequence number 11, comC in sequence number 13, comD in sequence number 15, comE in sequence number 17, comF in sequence number 19; and pilF in sequence number 21. Each of the above polynucleotide sequences includes a final three nucleotides representing a stop codon.

  Genes that use cDNA microarrays to characterize the regulation of NTHi genes during the development of receptive capacity (ie, the native ability of NTHi to incorporate foreign DNA that may enhance or expand its genetic diversity) In expression profiling studies, the com gene as well as the pil gene are upregulated during capacity development.

  Southern blot experiments using the pilA sequence as a probe with 13 low passage clinical NTHi OM isolates and cystic fibrosis recovered from patients who have undergone tympanotomy and tube insertion for chronic otitis media One clinical isolate recovered from the patient had one copy of pilA in their genome. A total of 14 isolates of these were assigned the following strain numbers: 86-028NP; 1728MEE; 1729MEE; 1714MEE; 214NP; 1236MEE; 165NP; 1060MEE; 1128MEE; 10548MEE; 3224A; 3185A, 1885MEEE and 27W11679INI .

  In this experiment, bacterial chromosomal DNA was isolated using the PUREGENE DNA isolation kit obtained from Gentra Systems (Minneapolis, Minn.), Digested with MfeI, and the digest was run on a 0.8% agarose gel. . DNA was transferred to the Nytran SuPerCharge membrane using the Turbo Blotter kit (Schleicher & Schuell, Keene, NH). The probe was made by PCR amplification of the 86-028NP pilA gene sequence code using primers 5'tgtgaactttccgcaaaaa (SEQ ID NO: 23) and 5'taataaaaaggaaaaatgaatga (SEQ ID NO: 24). The amplicon was purified using the QIA quick PCR purification kit (Qiagen Inc., Valencia, CA). 100 ng of purified PCR product was labeled with ECL Direct Nucleic Acid Labeling and Detection System (Amersham Biosciences UK Ltd., Little Chalfont, Bucks, UK) according to the manufacturer's instructions. The developed blot was exposed to Fuji Super Rx X-ray film (Fuji Photo Film Co., Tokyo, Japan).

  The PilA polypeptide of NTHi strain 86-028NP has an estimated and apparent molecular weight of approximately 14 kDa and contains an N-terminal methylated phenylalanine.

(Example 2)
NTHi strain 86-028NP formed classical aggregates in subsurface agar surface translocation and surface growth assays when grown under nutrient depletion conditions.

NTHi strain 86-028NP was grown on chocolate agar for 18-20 hours (37 ° C., 5% CO _{2 in a} humidified atmosphere) prior to all experiments. Subsequently, this organism was transformed into sBHI or 83% RPMI 1640 medium (Gibco BRL, Rockville, MD), sodium pyruvate (87.3 mM) (Gibco BRL), β-NAD (rich medium in which NTHi grows very well. 0.0087 mg / ml) (Sigma Chemical Co., St Louis, MO), HEME-histidine (0.0175 mg / ml) (Sigma), uracil (0.087 mg / ml) (Sigma), and inosine (1.75 mg) H./ml) (Sigma). One of the chemically defined media (Coleman et al., J. Clin. Micro., 41: 4408-4410, 2003) supporting growth of influenza was inoculated.

  Both agars were poured into one of two formats in a sterile 8-well chamber slide (Lab-tech, Naperville, IL) or into a sterile 35 mm glass petri dish (Fisher Scientific, location). When the glass slide is separated from the 8-well chamber slide, the agar remains in the chamber, thus allowing the use of the “bottom” surface of the agar for inoculation, which is the relative smoothness of this surface. It is optimal for assays of twitch contraction motility (Semmler et al., Microbiology, 145, 2863-2873, 1999 and Mattick, Ann. Rev. Microbiol., 56, 289-314, 2002). Agar molded in 8-well chamber slides was used to demonstrate the surface growth phenotype, but agar poured into petri dishes was used to demonstrate subsurface agar translocation (Semmler et al., Supra). While agar molded in 8-well chamber slides was used to show the growth phenotype of the surface of the agar. All assays were repeated a minimum of 3 times on another day.

Agar poured into a sterilized glass Petri dish was inoculated subsurface with a 0.5 μl suspension of NTHi grown as described above using a sterilized micropipette tip. Plates are observed after 24 hours of incubation (37 ° C., 5% CO ₂ ), and then an additional 24 before determining again the signs of bacterial translocation between the agar bottom surface and the glass petri dish. Hold at room temperature (25 ° C.) for hours.

  On sBHI medium, 24 hours after inoculation, NTHi was observed to grow in a small area (about 0.5 mm radius) around the inoculation site between the agar and the bottom of the glass petri dish. After another 24 hours, the growth pattern remained similar to that observed at 24 hours. After 24 hours incubation on chemically defined medium, NTHi growth was observed between the agar surface and the bottom of the glass petri dish at a distance of 2-5 mm from the inoculation site. The bacteria also aggregated into small colonies in a halo-like pattern around the inoculation site. After 48 hours, NTHi formed very differently aligned microcolonies, many of which occurred at distances greater than 5 mm from the inoculation site. The formation of micro-associates up to a distance of 5 mm from the original site of inoculation is a finding of a prominent feature of growth in chemically defined media, when strain 86-028NP is grown on rich agar Never seen.

  Chamber slides were suspended in 0.5 μl of NTHi grown on chocolate agar for 18-20 hours [suspended in pyrogen-free sterile saline (American Pharmaceutical Partners Inc., Schaumburg, IL) Or a single colony was punctured with a sterile toothpick for transfer to the surface of the agar.

  In sBHI medium, 30 minutes after inoculation, NTHi appeared in close association with the agar surface and grew in a sheet-like pattern. At 2.5 hours, about 80% to 90% of the surface area was covered with a thin sheet of bacteria. Also at this point, NTHi microaggregates began to appear. Six to seven hours after inoculation, the microaggregates were distinguishable with the naked eye and there were about 3-5 microaggregates per well. Furthermore, NTHi was still observed to grow as a sheet covering about 50% to 70% of the agar surface. 24 hours after inoculation, NTHi appeared as a large single colony at each inoculation site.

  Similar to what was observed on sBHI, in chemically defined media, 30 minutes after inoculation, NTHi appeared to grow in sheet form, but the bacterial density was on sBHI agar. Seemed much lower than that observed in. A large number of microaggregates were evident throughout the agar surface 2.5 hours after inoculation. In contrast to those shown, when NTHi was inoculated on sBHI agar, the microaggregates were larger in appearance and much denser. About 30-40 microaggregates could be seen on each well. In addition, at this point, there was a large area of NTHi sheet-like growth and about 80% of the surface area was covered by bacteria. At 6-7 hours after inoculation, the microaggregates were larger, denser and easily visible to the naked eye. Also, radial growth regions or halos are identified in Neisseria and Pseudomonas sp. Similar to the growth pattern described for (reference), it appeared to radiate outward from a large colony. By this period, most bacteria were aligned in small groups or microaggregates, with a very small percentage seen over the agar surface as sheets or monolayers. After 24 hours, there were large single colonies at each inoculation site, but there were also many small accompanying colonies that existed across the entire agar surface, including sites away from the inoculation point.

  Thus, NTHi strain 86-028NP, when grown under conditions of nutrient depletion, shows type IV pili expression P. pylori. Similar to that reported for aeruginosa (Semmler et al., Microbiology, 145 (10), 2863-2873, 1999), shows classical aggregate formation.

Example 3
The movement of individual NTHi cells between the coverslip and the smooth agar surface was followed by video microscopy. The cells migrated at about 0.42 μm / sec. This is because P. consistent with those reported for aeruginosa (Skerker and Berg, Proc. Natl. Acad., Sci. USA, 98, 6901-6904, 2001) and Neisseria gonorrhoeae (Merz et al., Nature, 207, 98-102, 2000). .

A platinum loop of NTHi strain 86-028NP that was grown on chocolate agar for 20 hours at 37 ° C. and 5% CO ₂ and then held at ambient temperature for an additional 24 hours was suspended in sterile water, The resulting 0.5 μl suspension was placed on a sterilized glass slide. To give contrast and thereby facilitate visualization, 0.5 μl trypan blue (0.4%, Sigma, St. Louis MO) was added to the bacterial suspension. The droplets were then covered with a sterilized cover glass and viewed through a light microscope (Axioscope 40, Zeiss, Thornwood, NY). Samples were observed at room temperature for about 15-20 minutes. Bacteria were easily observable, and not all but directional movements of some cells or cell microaggregates were noted. To stimulate activity, we added 0.5 μl of heme solution (1 mg / ml) (Sigma, St. Louis, MO) to one side of a sterilized cover glass. Twitch activity was demonstrated by the capture of both video [Video Otoscopy System Attached to VCR (MEDR _X Inc, Seminare, FL)] and further images to measure length and velocity of biased motion. .

  Individual cells, or microaggregates of cells, traveled a total linear distance of about 11.0 μm over 51 seconds (rate about 0.22 μm / sec). However, throughout the period of observation, the observed rate of twitch motion ranged from 0.14 μm / sec to 0.48 μm / sec.

Example 4
Type IV pili were visualized by negative staining and transmission electron microscopy.

An overnight culture of NTHi strain 86-028NP was inoculated onto sBHI and defined agar plates and incubated at 37 ° C., 5% CO ₂ for 2, 6, or 24 hours. In addition, the culture was inoculated into sBHI broth and defined broth and incubated for 2.5 or 5.5 hours. These latter time points indicate that the exponential and induction phases of growth have been entered, respectively. Bacteria were then negatively stained with Whatman filtered solution containing 2.0% ammonium acetate w / v (Sigma) and 2.0% ammonium molybdate w / v (Sigma) in sterile water (Bakaletz). Et al., Infect Immun, 1988 56: 331-5). Formvar-coated and carbon-coated copper grids (300 mesh) (Electron Microscience Sciences) were in contact with individual colonies grown on agar plates and then floated on the negative staining solution droplets. The broth growth culture was granulated, the bacteria were resuspended in sterile water and the grid was floated on an equal volume of bacterial suspension and negative stain. After 5 minutes, the grid was lightly wiped, air dried, and then a Hitachi Model H-600 transmission electron microscope fitted with a video monitor (Gatan, Inc., Pleasanton, Calif.) And a digital imaging system (Gatan, Inc.). Observed with.

  When NTHi was grown on sBHI, type IV pilus-like structures were not observed. Conversely, NTHi was found to express structures with a diameter of about 6-7 nm when grown under defined nutrient conditions. Many of these structures were also found free on the grid surface. There were about 5-6 pili per bacterial cell and they were in place and formed polarity.

(Example 5)
Mutants lacking expression of PilA were generated to further characterize the components of the structure observed when strain 86-028NP was grown in chemically defined media in alkaline conditions.

  The pilA gene from strain 86-028NP and about 1 kb of the 5 'and 3' sides of this gene were amplified by PCR and cloned into pGEM-T Easy (Promega) to determine its DNA sequence. As a result of PCR amplification, it was confirmed that there was no sequence change in the clones. Since there was no convenient restriction enzyme recognition site in the piA gene, the BamHI site was engineered into the gene using the Stratagene QuikChange Site-Directed Mutagenesis Kit. The resulting construct was linearized with BamHI and the gene was insert inactivated using the ΩKn-2 cassette (Perez-Casal et al., J. Bacteriol., 173: 2617-2624, 1991). The resulting construct was linearized and transformed into strain 86-028NP using the MIV method (Poje and Redfield, Herbert et al. (Eds.), Haemophilus influenzae Protocols, Humana Press Inc., Toronto, 2003. 57-70). Kanamycin resistant clones were selected to confirm insertional inactivation of the 86-028NP pilA gene in clones selected by Southern hybridization.

  When the piA mutant was evaluated for expression of type IV pili after growth under conditions that induced increased expression of type IV pili in the parental isolate (Example 4), No bound type IV pili or free type IV pili are observed, and the pilA gene product (and / or the piBCD gene product because the mutation is pilA that seems to disrupt the downstream gene product) It was confirmed that pili expression was required.

(Example 6)
In order to determine whether type IV pili are required for nasopharyngeal colonization and survival in the biofilm in the middle ear and / or the ability to form a biofilm, we Challenged 14 adult chinchillas both intranasally and transbully using either the parental strain 86-028NP or the syngeneic pilA mutant (Example 5). On the second, fifth, tenth, fifteenth and twentieth days after the challenge, nasopharyngeal lavage and upper puncture of the eardrum are performed, and one eardrum of the nose and middle ear is assigned to one group. Harvested from ˜2 chinchillas to determine the NTHi cfu in each of their anatomical sites. Both parent and pilA mutants were able to survive in the chinchilla host for 20 days. However, when assayed for adherent subpopulations in nasal mucosal tissue homogenates, both strains were present in equivalent amounts in lavage fluid and puncture fluid, but pilA mutants were homogenate recovered after day 5. 87% of similar nasal mucosa recovered from animals challenged with parental isolates were culture positive, while not present in 80% or below our detectability.

  Confocal microscopy was performed on the rapidly frozen tissue to determine if biofilm was present. Biomass formed by the pilA mutant in the middle ear was of a different characteristic from the well-constructed biofilm characteristic of the parental isolate. The data show that NTHi type IV pili play an important role in the disease process of OM.

(Example 7)
The pilA gene of 10 clinical isolates of NTHi was sequenced. The nucleotide and amino acid sequences from these isolates are shown below as follows: 1728 MEE in SEQ ID NO: 25 and SEQ ID NO: 26, 1729 MEE in SEQ ID NO: 27 and SEQ ID NO: 28, 3224A in SEQ ID NO: 29 and SEQ ID NO: 30 , 10548 MEE in SEQ ID NO: 31 and SEQ ID NO: 32, 1060 MEE in SEQ ID NO: 33 and SEQ ID NO: 34, 1885 MEE in SEQ ID NO: 35 and SEQ ID NO: 36, 1714 MEE in SEQ ID NO: 37 and SEQ ID NO: 38, 1236 MEE in SEQ ID NO: 39 and SEQ ID NO: 40 1128 MEE in SEQ ID NO: 41 and SEQ ID NO: 42, 214NP in SEQ ID NO: 43 and SEQ ID NO: 44. The alignment of these amino acid sequences with the amino acid sequence of the pilA polypeptide from Rd and the amino acid sequence of the pilA polypeptide from 86-028NP is shown in FIG.

  The pilA gene of all isolates encodes a 12-residue leader peptide that is largely invariant except that two isolates as well as replacing Q at position 6 with L in strain Rd. Mature PilA is predicted to contain 137 residues and a characteristic methylated phenylalanine at position +1. Tyrosine residues at positions +24 and +27 and thought to be involved in subunit-subunit interactions are highly conserved, as are the four Cys residues at positions +50, +60, +119, and +132. . Interestingly, the NTHi PilA protein appears to represent a new class of type IV pili. Leader peptides are longer than those characteristic of type IV pilins (typically 5-6 residues in length) and even shorter than typical IVb leader peptides (15-30 residues). At 137 residues, mature NTHi pilin is shorter than either class IVa pilin or IVb pilin (150 and 190 residues, respectively). Since NTHi PilA proteins are initiated with N-methylated phenylalanine, they are more likely class IVa pilins, but in electron micrographs, free NTHi type IV pili are antiparallel to each other. Due to the ability to self-associate by action, the phenotype that is more classically associated with class IVb pilins always appears in an associated bundle.

  Overall, the sequences are highly homologous with respect to NTHi PilA sequence diversity. See FIG. Due to protective immune predominance or attachment factor binding function, the surface is accessible and two areas of potentially important diversity when targeting vaccine development are positions 55-64 and 79 Present at ~ 87th position. Within the first region, it appears that there are two major variants between clinical isolates, while showing the majority (7 out of 11 isolates, 64%) , Sequence: characterized by NET / ITNCT / MGGGK, the other representing the minority (4 out of 11 isolates, 36%), characterized by the sequence: GKP / LST / SCSGGS. However, there are some additional minor variations at +57 and +61 in the majority grouping, and +57 and +59 for the minority grouping. The diversity shown at position +61 is currently only found in one isolate (strain # 1885) and there is a substitution of T to M. There appears to be two equally distributed variants between clinical NTHi isolates within the second focused region of difference (positions 79-87). The sequence ASVKTQSGG is present in 5 (about 45%) of 11 clinical isolates, while the sequence KSVTTSNGA is present in 6 (about 55%) of 11 clinical isolates. Exists.

  Overall, five of the seven isolates having a majority sequence at positions 55-64 also have a KSVTTSNGA motif in regions 79-87, and the remaining two singles The detachment has an ASVKTQSGG motif in this region. Three of the four remaining clinical isolates with minority sequences at positions 55-64 also have an ASVKTQSGG motif in regions 79-87, with only one isolate being This domain has the KSVTTSNGA sequence. Thus, depending on whether these are conservative substitutions and whether the sequence is present in the highly hydrophilic region of the surface available antigenicity index, and thereby becomes a target for vaccine development, They may or may not need to be included as a type IV pili-based component to elicit an immune response against NTHi.

(Example 8)
Four sequential syntheses to investigate the role of type IV pili in NTHi-induced OM and to determine whether antibodies from children with birth disease recognize type IV pili A peptide (which represents amino acids 21-137 of SEQ ID NO: 2 of mature PilA of NTHi strain 86-028NP) was synthesized and against a panel of pediatric sera and middle ear exudates obtained from children with OM Assayed by biosensor. Sera from children aged 2 months, 6 to 7 months or 18 to 19 months and 4 to 6 years of age with OM due to NTHi, as determined by the number of episodes of OM, and low incidence and Divided into high incidence groups.

  At present, antibodies in sera from children aged 2 months and 6 months to 7 months, either with a high or low incidence of OM, have been treated with either type IV pili peptide. It showed limited reactivity and had values of resonance units (RU) of 3-38 and 4-61, respectively. However, significant differences between these groups were seen with sera obtained from 18 to 19 months of age. Values obtained with sera from the low incidence 18- to 19-month group were 44 RU to 105 RU, but sera from the high incidence panel were five times higher for type IV pili peptide ( 81RU to 528RU). When a child was born and resolves OM at the age of 4-6 years, the reactivity to type IV pili peptide was again similar between the two incidence groups. To confirm that the reactivity observed here is specific to the disease caused by NTHi, S. Sera from children with OM due to pneumoniae were also assayed. In all cases, RU values of 16-120 were obtained for all type IV pili peptides. In order to assay for the presence of antibodies against type IV pili in exudates obtained from the middle ear, we also assayed those fluids with a biosensor. Exudates from children with OM due to Streptococcus were unreacted, but exudates collected from children with OM due to NTHi were highly reactive with type IV pili peptide. Collectively, this data strongly suggests that during the disease course of OM, NTHi type IV pili are expressed in vivo and that their structures are immunogenic.

Example 9
Identification of an immunogen that confers a broad cross-protective immune response against NTHi can be performed as follows.

(Synthesis of NTHi pilin peptide)
Panel of contiguous overlapping peptides and peptides from two focused regions of known diversity (see Example 6 above) to map the immunodominant and attachment factor binding domains of PilA Is synthesized. For example, 13 15-mer peptides with 5-residue overlap are synthesized to map the entire 137-residue mature pilin protein. The final C-terminal peptide is actually a 17mer spanning residues 121-137 to incorporate the final two amino acids of mature PilA. To fit the two described diversity regions, two peptide variants spanning residues 51-65 and two peptide variants spanning residues 79-95 are synthesized. In order to fully match this latter diversity region, the diversity region actually spans residues 79-87, so two peptides are made with varying lengths, one amino acid at the N-terminus. . Due to the additional residues, each of these latter two peptides is 16 mer in length. Thus, a total of 15 peptides are synthesized: 12 is a 15 mer, one is a 17 mer, and two are 16 mer peptides. This peptide is shown in Table 2 below, where the number of amino acid residues matches the amino acid of SEQ ID NO: 2.

（組換えＮＴＨｉピリン（ｒＰｉｌＡ）の作製）
組換えＰｉｌＡタンパク質（ｒＰｉｌＡ）を、アッセイにおいて完全なピリンサブユニットタンパク質を示すために使用するための、より容易に再生可能な産物として役立たせるために作製し得る。このようにするために、天然のピリンサブユニットの機能的な品質を所有するためにはｒＰｉｌＡが同様に適切に折りたたまれていることが重要であるので、４つのＣｙｓ残基を有したピリンを研究したＫｅｉｚｅｒら（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．、２７６：２４１８６−１４１９３、２００１）の公開されたプロトコルを利用する。手短かにいうと、先端を切り取ったピリンを操作し、ここで、最初の２８残基を、凝集を防止するために、Ｎ末端から取り除き、この先端を切り取ったピリンをさらに、構築物にＯｍｐＡリーダー配列の組み込みによって、ペリプラズムに運ばれるように操作する。この戦略を使用して、Ｋｅｉｚｅｒらは、インビトロでのアッセイにおいてそのレセプター（アシアロＧＭ１）に結合し得る組換え可溶性モノマーのＰ．ａｅｒｕｇｉｎｏｓａピリンタンパク質を作製して、異種チャレンジの前にこのペプチドを１５分送達した場合、マウスにおける罹患率および死亡率を減少させた。このＮＴＨｉ ＰｉｌＡの可溶性モノマー性の先を切り取った形態は、以下に記載した研究において有用である。

(Production of recombinant NTHi pilin (rPilA))
Recombinant PilA protein (rPilA) can be made to serve as a more easily reproducible product for use to represent the complete pilin subunit protein in the assay. To do this, it is important for rPilA to be similarly properly folded in order to possess the functional quality of the natural pilin subunit, so pilins with four Cys residues are Utilizes the published protocol of the studied Keizer et al. (J. Biol. Chem., 276: 24186-14193, 2001). Briefly, a truncated pilin was manipulated, where the first 28 residues were removed from the N-terminus to prevent aggregation, and this truncated pilin was further added to the OmpA leader in the construct. Manipulate it to be carried to the periplasm by incorporating the array. Using this strategy, Keizer et al. Have identified a recombinant soluble monomer, P.A., that can bind to its receptor (Asialo GM1) in an in vitro assay. Making an aeruginosa pilin protein and delivering this peptide for 15 minutes prior to the heterologous challenge reduced morbidity and mortality in mice. This soluble monomeric truncated form of NTHi PilA is useful in the studies described below.

(Mapping of immunodominant domain of PilA)
Peptides and native and recombinant PilA proteins are added to chinchilla and pediatric serum in acute and convalescent (in addition to middle ear fluids from chinchillas and children receiving experimental or innate OM due to NTHi) Used together in both our current sample collection or the collection is planned as part of another idea) by ELISA and also biosensor assays Map the immunodominant domain of PilA. Briefly, the relative amount of antibody that binds PilA peptide, rPilA and natural pilus to the surface of a 96-well microtiter plate or biosensor chip and then to each peptide in a serum or middle ear fluid sample. Assay.

  These studies identify regions that are relatively immunodominant over other of the pilin subunits when recognized by both chinchilla hosts and human children. The most N-terminal synthetic peptide is composed of very non-polar (hydrophobic) amino acids and therefore appears to be buried in the pilus fiber and is inaccessible to the antibody due to the fact that it is inaccessible to the 15-mer. The peptide is expected to serve as an internal negative control for the assays described herein. Normal pediatric sera and unstimulated chinchilla sera serve as negative serum controls, and middle ear lavage fluid collected from unstimulated animals is a negative control for exudates collected during NTHi infection of the middle ear. Used as.

(Mapping of attachment factor binding domain of PilA)
To map the eukaryotic binding domain of PilA, a competitive ELISA assay is performed, as well as the ability of synthetic pilin peptides to inhibit NTHi binding to eukaryotic cells in cell culture by confocal microscopy. For initial screening assays, appropriate eukaryotic target cells are grown in a 96 well microtiter dish. Cells are washed and then preincubated with synthetic pilin peptide, rPilA or native NTHi pili [0.2 μg in PBS] to promote NTHi strain 86-028NP (pirin expression to their eukaryotes) Their relative ability to block the binding of (grown under known conditions) was determined. The relative adherence of NTHi is determined by developing with tetramethylbenzidine (TMB) using a homologous complete NTHi OMP preparation and a polyclonal antiserum against HRP-binding protein A. For these appropriate assays, epithelial target cells [ie, chinchilla middle ear epithelial cells (CMEE), normal human bronchial / tracheal cells (NHuBr), human type II alveolar epithelial cell line (A549)], Epithelial target cells (CHO) that are not clinically relevant to which NTHi does not adhere as well as endothelial target cells [human umbilical vein endothelial cells (HUVEC)] are used.

  For peptides that exhibit inhibitory activity (typically the cut-off is more than 15% adhesion inhibition relative to the control), any dose dependence on the observed bacterial attachment blocking ability is determined. The interaction can be further evaluated by performing an adherent blockade assay using the Transwell system, where airway epithelial cells (CMEE and NHuBr) are grown at the air-fluid interface. These cells were isolated from the first synthetic peptide of interest (or isolated OMP P5 as a positive and negative control for NTHi surface proteins with or without an appropriate control, ie, adhesion, respectively. And P2, and rPilA) to try to block the available Tfp receptors, then wash them 5 times with fresh growth medium, followed by the inventor promoting Tfp expression Then inoculate about 2-5 × 10 7 NTHi grown under known conditions. The culture is washed to remove non-adherent bacteria, then fixed with methanol on ice for 5 minutes, air dried, rinsed with PBS, the membrane removed from the Transwell and examined by confocal microscopy. Place on cover glass to image. To detect adherent NTHi, using chinchilla hyperimmune anti-NTHi OMP serum and FITC-protein A, the interaction of NTHi with its epithelial target cells, or conversely, the putative attachment factor binding domain of PilA Demonstrating blockade of this interaction by the peptides shown.

(Selection of immunogen)
Based on the data obtained above, immunogenic peptides are selected based on both relative immunodominance as well as the ability to inhibit adherence of NTHi to respiratory epithelial target cells. Depending on the biochemical and structural characteristics of the region of interest, this peptide is produced as either a synthetic or recombinant peptide.

  The immunogenicity and protective efficacy of the PilA immunogen is first evaluated in the chinchilla animal model and human studies disclosed herein.

(Summary of Examples)
The foregoing evidence indicates that NTHi expresses functional type IV pili on their surface. The proteins encoded by these genes can be classified into H. pylori. It is known herein to be important for the ability to transform in influenza, and is also contemplated herein as important for biofilm formation by NTHi. Collectively, these observations indicate that NTHi appears to up-regulate the expression of type IV pili in a human host nutrient-restricted environment. Therefore, type IV pili are NTHi and H. It represents an excellent goal for vaccines and / or antibacterial strategies for pathogenic conditions caused by influenzae strains a, b, c, e and f.

Claims (1)

Invention described in the specification.

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